The immunogenicity of human-origin therapeutic antibodies are associated with V gene usage.

Therapeutic antibodies can elicit unwanted immune responses in a subset of patients, which leads to the production of anti-drug antibodies (ADA). Some of these ADAs have been reported to effect the pharmacokinetics, efficacy and/or safety of the therapeutic antibodies. The sequence diversity of antibodies are generated by VDJ recombination and mutagenesis. While the antibody generation process can create a large candidate pool for identifying high-affinity antibodies, it also could produce sequences that are foreign to the human immune system. However, it is not clear how VDJ recombination and mutagenesis impact the clinical ADA rate of therapeutic antibodies. In this study, we identified a positive correlation between the clinical ADA rate and the number of introduced mutations in the antibody sequences. We also found that the use of rare V alleles in human-origin antibody therapeutics is associated with higher risk of immunogenicity. The results suggest that antibody engineering projects should start with frameworks that contain commonly used V alleles and prioritize antibody candidates with low number of mutations to reduce the risk of immunogenicity.

An Anticancer Drug Cocktail of Three Kinase Inhibitors Improved Response to a Dendritic Cell-Based Cancer Vaccine.

Monocyte-derived dendritic cell (moDC)-based cancer therapies intended to elicit antitumor T-cell responses have limited efficacy in most clinical trials. However, potent and sustained antitumor activity in a limited number of patients highlights the therapeutic potential of moDCs. In vitro culture conditions used to generate moDCs can be inconsistent, and moDCs generated in vitro are less effective than natural DCs. On the basis of our study highlighting the ability for certain kinase inhibitors to enhance tumor antigenicity, we therefore screened kinase inhibitors for their ability to improve DC immunogenicity. We identified AKT inhibitor MK2206, DNA-PK inhibitor NU7441, and MEK inhibitor trametinib as the compounds most effective at modulating moDC immunogenicity. The combination of these drugs, referred to as MKNUTRA, enhanced moDC activity over treatment with individual drugs while exhibiting minimal toxicity. An evaluation of 335 activation and T-cell-suppressive surface proteins on moDCs revealed that MKNUTRA treatment more effectively matured cells and reduced the expression of tolerogenic proteins as compared with control moDCs. MKNUTRA treatment imparted to ICT107, a glioblastoma (GBM) DC-based vaccine that has completed phase II trials, an increased ability to stimulate patient-derived autologous CD8+ T cells against the brain tumor antigens IL13Rα2(345-354) and TRP2(180-188) In vivo, treating ICT107 with MKNUTRA, prior to injection into mice with an established GBM tumor, reduced tumor growth kinetics. This response was associated with an increased frequency of tumor-reactive lymphocytes within tumors and in peripheral tissues. These studies broaden the application of targeted anticancer drugs and highlight their ability to increase moDC immunogenicity.

Clinical Manifestations of an Anti-Drug Antibody Response: Autoimmune Reactions.

Antibodies can be generated against a therapeutic protein upon administration to human subjects. When the therapeutic protein closely mimics one of the subject's endogenous proteins, those antibodies might bind to the endogenous protein in addition to the therapeutic protein. This scenario results when tolerance to the endogenous protein is broken. The consequences of breaking tolerance include an autoimmune response where antibodies are generated against the endogenous protein. These autoantibodies could have significant clinical relevance depending on several factors, including the redundancy of action of the endogenous protein as well as the concentration, binding affinity, and neutralizing potential of the antibodies. The consequences of a therapeutic-protein-induced autoimmune reaction can be challenging to manage as the stimulus for further perpetuation of the immune response can shift from the therapeutic protein to the endogenous protein. The potential for inducing an autoimmune response is one of the reasons that the immune response to a therapeutic protein should be monitored if it persists through the end of the study.

Strategic characterization of anti-drug antibody responses for the assessment of clinical relevance and impact.

All therapeutic proteins have the potential to induce anti-drug antibodies (ADA). Clinically relevant ADA can impact efficacy and/or safety of a biological therapeutic. Immunogenicity assessment strategy evaluates binding and neutralizing ADA, and the need for additional characterization (e.g., epitope, titer and so on) is determined using a risk-based approach. The choice of characterization assays depends on the type, application and immunogenicity of the therapeutic. ADA characterization can impact the interpretation of the risk profile of a given therapeutic, and offers insight into opportunities for risk mitigation and management. This article describes common ADA characterization methods. Strategic assessment and characterization of clinically relevant ADA are discussed, in order to support clinical options for safe and effective patient care and disease management.

Development of a biosensor-based immunogenicity assay capable of blocking soluble drug target interference.

As with other protein therapeutics, trebananib (AMG 386), an investigational peptide Fc-fusion protein ("peptibody") that inhibits angiogenesis by neutralizing the interaction of angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) with the Tie2 receptor, has the potential to trigger an immune response in cancer patients treated with the therapeutic. An electrochemiluminescence bridging anti-drug antibody (ADA) assay that was utilized to support early-phase clinical trials in the development of trebananib was found to lack adequate sensitivity and drug tolerance in later-phase clinical studies when higher doses of trebananib were administered. Therefore, we developed a surface plasmon resonance (SPR) immunoassay method utilizing a secondary confirmatory detector antibody (goat anti-human IgG F[ab']2) known to cross-react with human IgG and IgM to better assess the potential impact of immunogenicity on the pharmacokinetics, pharmacodynamics, and toxicity of trebananib. The SPR method was more sensitive than the electrochemiluminescence bridging assay because of signal amplification from the confirmatory binding of the detector antibody; drug tolerance was improved since antibody binding avidity does not affect detection on this platform. Despite the inability of the confirmatory detector antibody to bind angiopoietins in protein-free buffer, false-positive ADA results were generated from patient serum samples containing Ang1 and Ang2 through an apparently specific binding between the angiopoietins and the confirmatory detector antibody, likely mediated by the interaction of the angiopoietins with serum immunoglobulins. Addition to the sample diluent of a human antibody that specifically binds to Ang1 and Ang2 with high affinity resulted in a complete block of angiopoietin interference without affecting ADA detection. This biosensor-based assay provides a reliable method for assessing immunogenicity in phase 3 clinical trials.

Immunogenicity testing strategy and bioanalytical assays for antibody-drug conjugates.

BACKGROUND: Immunogenicity testing is an important component of clinical development for large-molecule biotherapeutics. New complex types of large molecules, such as antibody-drug conjugates (ADCs), require careful evaluation of the testing strategy and bioanalytical assays used to monitor the development of antitherapeutic antibodies. RESULTS: An electrochemiluminescence-based immunoassay for the detection and epitope characterization of anti-ADC antibodies was validated. Using this assay format, antibodies directed against the monoclonal antibody and linker-drug components of the ADC were successfully detected in a multiple-dose rat toxicity study. CONCLUSION: Immunogenicity assays incorporating epitope determination may provide additional information about the characteristics of induced antitherapeutic antibodies, including the magnitude and timing of the various types of antibody responses.

Stratification of antibody-positive subjects by antibody level reveals an impact of immunogenicity on pharmacokinetics.

The availability of highly sensitive immunoassays enables the detection of antidrug antibody (ADA) responses of various concentrations and affinities. The analysis of the impact of antibody status on drug pharmacokinetics (PK) is confounded by the presence of low-affinity or low-concentration antibody responses within the dataset. In a phase 2 clinical trial, a large proportion of subjects (45%) developed ADA following weekly dosing with AMG 317, a fully human monoclonal antibody therapeutic. The antibody responses displayed a wide range of relative concentrations (30 ng/mL to >13 µg/mL) and peaked at various times during the study. To evaluate the impact of immunogenicity on PK, AMG 317 concentration data were analyzed following stratification by dose group, time point, antibody status (positive or negative), and antibody level (relative concentration). With dose group as a stratifying variable, a moderate reduction in AMG 317 levels (<50%) was observed in antibody-positive subjects when compared to antibody-negative subjects, but the difference was not statistically significant in all dose groups. The most significant reduction in AMG 317 levels was revealed when antibody data was stratified by both time point and antibody level. In general, high ADA concentrations (>500 ng/mL) and later time points (week 12) were associated with significantly (up to 97%) lower trough AMG 317 concentrations. The use of quasi-quantitative antibody data and appropriate statistical methods was critical for the most comprehensive evaluation of the impact of immunogenicity on PK.

Immunogenicity assessment in non-clinical studies.

Recent ICH S6 guidance on preclinical safety evaluation of biotechnology derived biopharmaceuticals indicates that testing for anti-drug antibodies is not always required to establish the safety of a protein therapeutic. Most human protein therapeutics will induce a rapid and robust anti-drug antibody response in preclinical studies and the presence of high levels of circulating drug complicates the detection of anti-drug antibodies. The presence of anti-drug antibodies in preclinical studies does not predict if a protein therapeutic will be immunogenic in the clinic. When testing for anti-drug antibodies is warranted, there are a variety of analytical procedures that can be utilized, although each of these methods has advantages as well as limitations. Immunoassays can be used to identify if antibodies are present that bind to the therapeutic, and when necessary, biological assays can be used to identify if those antibodies neutralize the effect of the therapeutic. Under certain circumstances including intravenous dosing of a mAb therapeutic, anti-drug antibodies can form large immune complexes that can result in a safety issue. The value of immunogenicity data in preclinical studies is to aid in interpretation of other study data when necessary.

Development and characterization of a human antibody reference panel against erythropoietin suitable for the standardization of ESA immunogenicity testing.

Recombinant human erythropoietin (EPO) has been used therapeutically for more than two decades in the treatment of anemia. Although EPO is generally well tolerated, in rare cases, patients have developed anti-EPO antibodies that can negatively impact safety and efficacy. Therefore, the detection of antibodies against EPO is a regulatory requirement during clinical development and post-approval. Although it is a rare phenomenon, antibody-mediated pure red cell aplasia (PRCA) is a serious complication than can result from antibodies that develop and neutralize EPO as well as endogenous erythropoietin. Currently, there are no universally accepted analytical methods to detect the full repertoire of binding and neutralizing anti-EPO antibodies. A number of different methods that differ in terms of antibodies detected and assay sensitivities are used by different manufacturers. There is also a lack of antibody reference reagents, and therefore no consistent basis for detecting and measuring anti-EPO antibodies. Reference reagents, with established ranges, are essential to monitor the safety and efficacy of all erythropoiesis-stimulating agents (ESAs) structurally related to human erythropoietin. This is the first report of the development and characterization of a panel of fully human antibodies against EPO suitable as reference reagents. The characteristics of antibodies within the panel were selected based on the prevalence of non-neutralizing IgG and IgM antibodies in non-PRCA patients and neutralizing IgG antibodies, including IgG1 and IgG4, in antibody-mediated PRCA subjects. The reference panel includes antibodies of high- and low-affinity with binding specificity to neutralizing and non-neutralizing erythropoietin epitopes. The subclass of human antibodies in this reference panel includes an IgG1, IgG2, and IgG4, as well as an IgM isotype. This antibody panel could help select appropriate immunogenicity assays, guide validation, and monitor assay performance. Further, this human anti-ESA antibody panel may help set the limits of each assay platform in terms of the full repertoire of the anti-ESA antibodies, and may facilitate standardization of ESA immunogenicity reporting across assay platforms.

Epitope characterization of pre-existing and developing antibodies to an aglycosylated monoclonal antibody therapeutic of G1m17,1 allotype.

Allotypes of IgG1 molecules can influence the immunogenicity of therapeutic monoclonal antibodies and may account for the presence of some pre-existing antibodies. An electrochemiluminescent (ECL) bridging immunoassay was used to characterize the binding epitopes of anti-therapeutic antibodies (ATAs) in a Phase 1 single ascending dose clinical trial of a therapeutic aglycosylated IgG1monoclonal antibody (mAb). There was no evidence for ATAs specific for a possible neo-epitope created due to the lack of glycosylation. ATAs that developed post-treatment were specific for the F(ab')2, whereas, pre-existing ATAs were specific to the Fc region. Further characterization of the pre-existing ATAs identified the specific epitope to be the G1m1 allotype determinant in the Fc of the therapeutic. A novel competitive bridging assay was developed to verify that serum IgG1 from subjects with pre-existing anti-G1m1 antibodies was homozygous for the antithetical allotype (G1m3). The endogenous G1m allotype of all subjects was assessed and correlation to ATA incidence and adverse events was evaluated. Interestingly, the pre-existing anti-allotype antibody in subjects persisted but was not augmented after dosing, indicating the lack of a secondary immune response to this epitope. These studies indicate the relationship of the therapeutic allotype and the corresponding allotype of subjects is an important component to further understand the impact of immunogenicity on the safety and efficacy of therapeutic antibodies.

Detection of anti-ESA antibodies in human samples from PRCA and non-PRCA patients: an immunoassay platform comparison.

BACKGROUND: The immunological methods for detecting antibodies to erythropoiesis-stimulating agents (ESAs) differ in assay sensitivity. However, this parameter, routinely determined in clinical assays using a high-affinity non-human polyclonal antibody, gives a one-dimensional assessment of antibody detection. We compare three widely used immunological methods and evaluate the ability of each to detect mature human antibodies and human antibodies characteristic of an early immune response. METHODS: The detection of anti-ESA antibodies was compared between a radioimmunoprecipitation (RIP) assay, an electrochemiluminescence (ECL) bridging enzyme-linked immunosorbent assay and a surface plasmon resonance (SPR)-based immunoassay. All three methods were validated for sensitivity, specificity and precision. Specimens from clinical studies or post market testing were categorized as pure red cell aplasia (PRCA) or non-PRCA and then analyzed in each method. RESULTS: Among the antibody-mediated PRCA samples, which contain high affinity neutralizing antibodies, there was strong correlation between all methods. The results from non-PRCA sample analysis show high correlation between RIP and ECL methods; however, differences between the SPR immunoassay and the ECL and RIP were demonstrated. The samples that scored positive in the SPR immunoassay and negative by RIP and ECL were characterized to be of low antibody concentration, contained a high percentage of rapidly dissociating antibodies, or were antibodies of the IgM isotype. CONCLUSIONS: All three immunological methods are appropriate for detection of antibodies associated with antibody-mediated PRCA. However, the SPR immunoassay platform detected an early, low affinity IgG and IgM antibody response as well as detected and characterized a pathogenic antibody response associated with antibody-mediated PRCA.

Immunogenicity of panitumumab in combination chemotherapy clinical trials.

BACKGROUND: Panitumumab is a fully human antibody against the epidermal growth factor receptor that is indicated for the treatment of metastatic colorectal cancer (mCRC) after disease progression on standard chemotherapy. The purpose of this analysis was to examine the immunogenicity of panitumumab and to evaluate the effect of anti-panitumumab antibodies on pharmacokinetic and safety profiles in patients with mCRC receiving panitumumab in combination with oxaliplatin- or irinotecan-based chemotherapies. METHODS: Three validated assays (two screening immunoassays and a neutralizing antibody bioassay) were used to detect the presence of anti-panitumumab antibodies in serum samples collected from patients enrolled in four panitumumab combination chemotherapy clinical trials. The impact of anti-panitumumab antibodies on pharmacokinetic and safety profiles was analyzed using population pharmacokinetic analysis and descriptive statistics, respectively. RESULTS: Of 1124 patients treated with panitumumab in combination with oxaliplatin- or irinotecan-based chemotherapy with postbaseline samples available for testing, 20 (1.8%) patients developed binding antibodies and 2 (0.2%) developed neutralizing antibodies. The incidence of anti-panitumumab antibodies was similar in patients with tumors expressing wild-type or mutant KRAS and in patients receiving oxaliplatin- or irinotecan-based chemotherapies. No evidence of an altered pharmacokinetic or safety profile was found in patients who tested positive for anti-panitumumab antibodies. CONCLUSIONS: The immunogenicity of panitumumab in the combination chemotherapy setting was infrequent and similar to the immunogenicity observed in the monotherapy setting. Panitumumab immunogenicity did not appear to alter pharmacokinetic or safety profiles. This low rate of immunogenicity may be attributed to the fully human nature of panitumumab.

Strategy to confirm the presence of anti-erythropoietin neutralizing antibodies in human serum.

Functional cell-based assays are the preferred method to test for the presence of anti-rHuEPO neutralizing antibodies (NAbs). However, due to the unpredictable nature of test serum matrix effects on cell-based assays, confirmatory assays are essential for verifying NAb positive results observed during the course of sample testing. The cell-based assay used for the detection of NAbs described by Wei et al. [1] used 32D-EPOR cells, a murine myeloid cell line transfected with the human EPO receptor (EPOR). The 32D-EPOR cell line responded to either rHuEPO or murine interleukin 3 (mIL-3) with proliferation. NAbs were expected to only inhibit rHuEPO-induced cell proliferation and not mIL-3 induced proliferation. Due to reliance on proliferation, the results from this cell-based assay can be confounded by the presence of non-antibody inhibitory serum factors. This paper describes a strategy for confirming that the inhibition of rHuEPO-induced proliferation in a cell-based assay is only attributable to NAbs. The strategy of antibody depletion uses a resin mixture composed of Protein G Sepharose and Protein L Sepharose (Protein G/L resin) to significantly reduce the concentration of immunoglobulins of IgG, IgM and IgA isotypes from human serum prior to testing in the cell-based assay. If the reduction in immunoglobulins in a serum sample corresponds to a reduction in inhibition of EPO-induced proliferation, it would infer that EPO neutralizing activity is antibody-mediated and not due to non-antibody inhibitory serum factors.

Impact of matrix-associated soluble factors on the specificity of the immunogenicity assessment.

BACKGROUND: Specificity and sensitivity are essential in assays for immunogenicity assessment of biotherapeutics. Nonspecific interactions from excess therapeutic or anti-therapeutic antibody, soluble ligands (e.g., target receptor), or serum proteins associated with autoimmune conditions (e.g., rheumatoid factor) in samples can impact the detection of a true anti-therapeutic response. RESULTS: Electrochemiluminescence-based bridging assay formats could eliminate the interference due to rheumatoid factor with no pretreatment with Melon Gel™ or aggregated IgG. The interference due to soluble factors was not platform specific for the four therapeutics evaluated in this study. CONCLUSION: Melon Gel pretreatment and avidin high-bind (Meso Scale Discovery) plates can effectively reduce interference due to rheumatoid factor in ELISA- and electrochemiluminescence-based assays, respectively. Excess levels of therapeutic and anti-therapeutic antibodies in bridging assays can impact assay specificity.

Identification and inhibition of drug target interference in immunogenicity assays.

A well-designed anti-drug antibody (ADA) immunoassay is critical for appropriately monitoring the immunogenicity profile of a therapeutic protein during its development. AMG 386 is a peptide-Fc fusion protein that inhibits angiogenesis by preventing the interaction of angiopoietins with the Tie2 receptor. In bridging immunoassays for ADA, interference by the drug target, present in the assay sample, can result in false positive antibody detection. We used a statistical design-of-experiments approach to identify angiopoietin interference in bridging immunoassays of anti-AMG 386 antibodies. We also demonstrated that a high-affinity monoclonal antibody, directed against an epitope on angiopoietin that competes with AMG 386 binding, could inhibit the angiopoietin interference while preserving the detection of ADA. This report describes the development and validation of methodologies for evaluating and addressing drug target interference in bioanalytical assays that involve interactions between drug, ADA, immune complexes, and drug target.

Rheumatoid factor interference in immunogenicity assays for human monoclonal antibody therapeutics.

Rheumatoid factors (RFs) are endogenous human antibodies that bind to human gamma globulins. RFs demonstrate preferential binding to aggregated gamma globulins and are involved in the clearing mechanism of immune complexes. Immunoassays designed to measure human anti-human antibodies (HAHA) after administration of monoclonal antibody therapeutics are thus vulnerable to interference from RFs. When using a sensitive electrochemiluminescent (ECL) bridging immunoassay, samples from subjects with rheumatoid arthritis demonstrated much higher baseline reactivity than healthy subjects. Interference was found to be dependent on the aggregation state of the therapeutic antibody that had been conjugated with the detection reagent (ruthenium). Size exclusion high performance liquid chromatography (SE-HPLC) demonstrated that of the total integrated peaks, as little as 0.55% high molecular weight aggregates (>600kDa) were sufficient to cause increased reactivity. Stability studies of the ruthenium and biotin conjugated therapeutic antibody indicated that storage time, temperature and buffer formulation were critical in maintaining the integrity of the reagents. Through careful SE-HPLC monitoring we were able to choose appropriate storage and buffer conditions which led to a reduction in the false reactivity rate in therapeutic-naïve serum from a rheumatoid arthritis population.

Assessment of immunogenicity of romiplostim in clinical studies with ITP subjects.

Romiplostim is an Fc-peptide fusion protein that activates intracellular transcriptional pathways via the thrombopoietin (TPO) receptor leading to increased platelet production. Romiplostim has been engineered to have no amino acid sequence homology to endogenous TPO. Recombinant protein therapeutics can be at a risk of development of an antibody response that can impact efficacy and safety. Hence, a strategy to detect potential antibody formation to the drug and to related endogenous molecules can be useful. The immunogenicity assessment strategy involved both the detection and characterization of binding and neutralizing antibodies. The method for detection was based on a surface plasmon resonance biosensor platform using the Biacore 3000. Samples that tested positive for binding antibodies in the Biacore immunoassay were then tested in a neutralization assay. Serum samples from 225 subjects with immune thrombocytopenic purpura (ITP) dosed with romiplostim and 45 ITP subjects dosed with placebo were tested for romiplostim and TPO antibodies. Prior to romiplostim treatment, 17 subjects (7%) tested romiplostim antibody positive and 12 subjects (5%) tested TPO antibody positive for pre-existing binding antibodies. After romiplostim exposure, 11% of the subjects exhibited binding antibodies against romiplostim and 5% of the subjects with ITP showed binding antibodies against TPO. The antibodies against romiplostim did not cross-react with TPO and vice versa. No cases of anti-TPO neutralizing antibodies were detected in romiplostim-treated subjects. The incidence of anti-romiplostim neutralizing antibodies to romiplostim was 0.4% (one subject); this subject tested negative at the time of follow-up 4 months later. No impact on platelet profiles were apparent in subjects that had antibodies to romiplostim to date. In summary, administration of romiplostim in ITP subjects resulted in the development of a binding antibody response against romiplostim and TPO ligand. One subject developed a neutralizing antibody response to romiplostim that impacted the platelet counts of this subject. No neutralizing antibodies to endogenous TPO were observed.

Comparing exponentially weighted moving average and run rules in process control of semiquantitative immunogenicity immunoassays.

The immunogenicity immunoassay validation process ensures development of a robust, reproducible method. However, no matter how well developed, validated, and maintained a method is, in the course of running a large number of samples over time, it is not uncommon to see bad reagents, poorly calibrated equipment, personnel errors, or other unknown and unpredictable factors that have an impact in the performance of the method and quality of the sample results. The immunogenicity immunoassay thus needs to be closely monitored with an internal statistical quality control process overtime to ensure a consistent and reliable output. The statistical process control has been widely applied to monitor manufacturing processes and in clinical laboratories. Its application to immunogenicity immunoassays is relatively novel. Limited guidance is available to implement the process to monitor semiquantitative immunogenicity immunoassay performance. Here, we have performed a suitability evaluation for process control charts with actual laboratory data from three immunogenicity immunoassay methods each utilizing a different technology platform. Additionally, a panel of prepared samples designed to assess long-term method performance were periodically evaluated for over a year. Finally, we make recommendations for an internal quality control process based on the results of these evaluations.

A step-wise approach for transfer of immunogenicity assays during clinical drug development.

We designed a three-step statistical approach to transfer bioanalytical assays (ELISA and Biacore) which evaluates the (1) average equivalence between the two labs (2) concordance in individual sample results between the two labs, and (3) long-term stability of assay performance. Each experimental design evaluated the contribution of four critical variables to the overall variability. Two lots of each variable were examined in a controlled experiment. The variables tested for ELISA were analyst, plate washer, biotinylated-therapeutic protein, and streptavidin-horseradish peroxidase; and for Biacore were analyst, instrument, chip lot, and conjugation chemistry reagent lots. Equivalence in the mean signal to noise (S/N) or mean relative units (RU) between the two labs was established through statistical evaluation of the assay performance characteristics across multiple assay variables. Concordance between the two labs in the individual sample results was subsequently verified both quantitatively and qualitatively. The long-term maintenance of assay stability was monitored by performance testing of a predefined set of samples which were prepared in sufficient quantities to last several years. The process of method validation for biomarker testing in clinical trials is to analyze the variability of the assay performance. However, different factors contribute to this variability and need to be evaluated when the method is transferred to another site/lab. Lack of understanding the critical variables can potentially result in unexpected problems and delays. The three-step statistical approach of assay transfer provides a robust process for transferring complex biological assays.

The development and validation of a sensitive, dual-flow cell, SPR-based biosensor immunoassay for the detection, semi-quantitation, and characterization of antibodies to darbepoetin alfa and epoetin alfa in human serum.

A surface plasmon resonance (SPR)-based biosensor immunoassay was developed and validated using the Biacore 3000 instrument to detect, semi-quantitate, and characterize serum antibodies against darbepoetin alfa (Aranesp) and epoetin alfa (EPOGEN). In this sensitive, dual-flow cell assay, epoetin alfa and darbepoetin alfa are covalently immobilized onto consecutive flow cells of a carboxymethyl dextran-coated sensor chip. Diluted human serum samples are injected sequentially over both surfaces. The binding of serum antibodies to the immobilized proteins are detected and recorded in real time based on the principles of SPR. Furthermore, antibody binding is confirmed with a secondary anti-human immunoglobulin antibody. Positive samples are further characterized to determine the relative concentration of the antibodies using an affinity-purified, rabbit anti-epoetin alfa antibody as a reference control. The assay can detect 80ng/ml and 100ng/ml of antibody to epoetin alfa and darbepoetin alfa, respectively. The dynamic range of the assay is from 0.078microg/ml to 10microg/ml using a rabbit antibody with demonstrated accuracy and intra- and inter-assay precision. Approximately 80 serum samples can be analyzed on each sensor chip while maintaining a stable baseline and consistent immunological reactivity. The analysis of serum samples from subjects administered with epoetin alfa or darbepoetin alfa provided evidence that the assay can detect varying concentrations of antibodies of different off rates, isotypes, and IgG subclasses.